Download Dynabeads® for protein complex isolation

Application Note
Dynabeads for protein
complex isolation
®
Studying the way proteins function and interact is an exciting area of research. By looking
at protein structures, researchers can identify how cofactors and other molecules influence
enzymatic or protein activity.
Isolating complete protein complexes has always been problematic because the weak,
ionic interactions between proteins can easily be destroyed by mechanical stress. In this
application note, we discuss how to pull down intact protein complexes using Dynabeads®
magnetic separation technology.
Application Note
Traditional methods
Existing techniques for isolating proteins include pulldown with
Sepharose® beads and spin columns, but these methods have
disadvantages. With Dynabeads®, you can skip unnecessary steps
that can cause your protein complexes to dissolve, including:
••
••
••
••
Exposure to large surfaces
Mechanical strain (e.g., centrifugation)
Dilution
Excessive handling (preclearing)
Although some researchers choose to preclear using Sepharose®
beads, there can be nonspecific binding interactions that can
contaminate the final product.
The Dynabeads® method
Add your specific antibody or interacting protein with tags to
Dynabeads®, then immunoprecipitate your protein of interest.
Once the beads are exposed to a magnet, they are efficiently
drawn to the tube wall, taking only your protein complex
with them (Figure 1). As the process is gentle, yet very quick,
complexes remain intact and functional. The complexes can be
resuspended in a small volume ready for downstream analysis
with mass spectrometry, gels, etc. Advantages of Dynabeads®
include:
••
••
••
••
Quick and easy pulldown of intact, functional complexes
No time-consuming preparation steps
Only isolate the proteins you want
Can be adapted for high-throughput applications
There is a range of Dynabeads® to choose from depending
on your antibody or protein. Dynabeads® ready-coated with
Protein A or Protein G are available, or you can couple your own
antibodies or proteins with tags to beads such as Dynabeads®
M-270 Epoxy or Dynabeads® Streptavidin.
Below are some examples of how Dynabeads® have been
used in published articles to pull down protein complexes.
Featured papers
Cristea et al. used a single green fluorescent protein (GFP)
tag to visualize proteins and their interactions in live cells
using immunoaffinity purification with Dynabeads® M-270 Epoxy
and anti-GFP antibodies. They showed that purification was rapid
and efficient, and that protein complexes were in their original
state with minimal nonspecific interactions. They predict that
the method will help researchers understand many cellular
processes.
Dynabeads® M-270 Epoxy were incubated with anti-GFP
polyclonal antibodies or IgG and added to the soluble cell lysate
fraction. These antibody-coated Dynabeads® bound to the target
proteins and when the tube was placed in a magnet, were drawn
to the tube wall. After washing, the isolated protein complexes
were eluted from the beads, frozen with liquid nitrogen, and left
to dry overnight by vacuum centrifugation. The pellet was put
in SDS-PAGE buffer, separated on a precast 1-D gel before being
stained with Coomassie® blue, prior to mass spectrometry.
This protocol can be applied to sample materials such as
viruses, bacteria, yeast, mammalian tissue, cultured cells, and
mice.
Option 1:
Use bead-bound
targets for
further studies
Incubate Dynabeads®
with the sample to form
target-based complexes
Figure 1—Isolation of protein complexes using Dynabeads®.
2
Isolate complexes
with magnet and
remove supernatant
Option 2:
Elute target
off beads for
further studies
Devos, et al. wanted to study the nuclear pore complex (NPC),
the gate that mediates traffic of macromolecules across the nuclear
envelope. They used computational and biochemical means to
analyze the seven proteins in one of the subcomplexes in the
NPC. As part of the study, they performed proteolytic mapping of
domain boundaries and loop locations in the seven yeast extract
nups, the set of proteins that make up the NPC. The Protein
A–tagged nups were bound to Dynabeads® M-270 Epoxy with
proteolytically resistant tags and purified from yeast extracts with
magnetic separation. Endoproteinases were added to hydrolyze
peptide bonds and any proteolytic fragments on the beads were
separated by SDS-PAGE. Cleavage sites were determined by amino-terminal Edman sequencing or by estimating the molecular
weight of the fragments.
Khalili, et al. were investigating prion diseases and how the
normal cellular prion protein PrP is converted into an abnormal
isoform (PrPSc) in prion-diseased brains. They produced
monoclonal antibodies (mAbs) for immunoprecipitation (IP) of
PrPSc and the glycoforms from diseased and normal brain homogenates. Direct IP was performed using mAbs crosslinked to
Dynabeads®, mouse IgG on Dynabeads® Protein A, or biotinylated
antibodies with Dynabeads® M-280 Streptavidin. Indirect IP was
used to capture antibody-antigen complexes with anti-PrP mAbs
IgG and Dynabeads® Protein G. They demonstrated that the
differentially glycosylated native PrPSc are closely associated and
immunoprecipitate together.
Suzuki, et al. developed an in vitro pulldown assay that
uses in vitro biotinylated proteins rather than tagged proteins as
pulldown drivers with Dynabeads® Streptavidin. They synthesized
the biotinylated proteins by in vitro transcription-translation
with biotin-lysine transfer RNA. They identified the following
advantages:
•• No need to prepare plasmids for the pulldown drivers
•• Expressed proteins are likely to be soluble
•• Random position of biotin-labelled lysine residues throughout
the protein mean that the fused tag does not interfere with
interactions
•• Biotinylated proteins are functional in many cases and maintain their native conformations
Products
Product
Volume
Cat. no.
1 ml
100.01D
5 ml
100.02D
1 ml
100.03D
5 ml
100.04D
60 mg
143.01D
300 mg
143.02D
2 ml (30 mg/ml)
142.03D
10 ml (30 mg/ml)
142.04
10 ml (100 mg/ml)
301.01
2 ml
112.05D
10 ml
112.06D
100 ml
602.10
2 ml
656.01
10 ml
656.02
100 ml
656.03
2 ml
112.01D
10 ml
112.02D
2 ml
112.03D
10 ml
112.04D
Dynabeads® Protein A
Dynabeads® Protein G
Dynabeads® M-270 Epoxy
Dynabeads® M-280 Tosylactivated
Dynabeads® M-280 Streptavidin
Dynabeads® MyOne Streptavidin T1
Dynabeads® M-280 Sheep anti-Mouse IgG
Dynabeads® M-280 Sheep anti-Rabbit IgG
Choose for:
For use with:
• Human IgG 1, 2, 4
• Mouse IgG2a, 2b, 3
• Rat IgG2c
• Bovine IgG2
• Canine IgG
• Goat IgG2
• Guinea Pig IgG
• Monkey IgG
• Porcine IgG
• Rabbit IgG
• Sheep IgG2
For use with:
• Human IgG 1, 2, 3, 4
• Mouse IgG1, 2a, 2b, 3
• Rat IgG2a, 2c
• Bovine IgG1, 2
• Goat IgG1, 2
• Guinea Pig IgG
• Horse IgG
• Monkey IgG
• Porcine IgG
• Rabbit IgG
• Sheep IgG1, 2
For gentle binding of structurally intact and active peptides,
proteins, and enzymes—hydrophilic surface.
For easy coupling of antibodies for affinity capture of
proteins—hydrophobic surface.
For use with biotinylated proteins.
For general protein purification, sequence-specific DNA/RNA
capture, and biopanning. Needs BSA blocking.
Ideal for manual or automated protocols—low-charged and
neutral beads optimal for proteins, peptides, and antibodies.
For use with mouse IgG1, IgG2a, and IgG 2b, not IgG3. Fc
reactive.
For use with any rabbit IgG antibody.
3
www.invitrogen.com
Application Note
Featured papers:
1. Cristea, I.M. et al. (2005) Fluorescent Proteins as Proteomic Probes. Molecular & Cellular Proteomics 4(12): 1933–1941.
2. Devos, D. et al. (2004) Components of coated vesicles and nuclear pore complexes share a common molecular architecture. PLoS Biol 2(12): e380.
3. Khalili-Shizari, et al. (2005) PrP glycoforms are associated in a strain-specific ratio in native PrPSc. J General Virology 86: 2635–2644.
4. Suzuki, H. et al. (2004) In vitro pull-down assay without expression constructs. BioTechniques 37 (6): 918–919.
Other relevant articles:
1. Aartsen, W.M. et al. (2006) Mpp4 recruits Psd95 and Veli3 towards the photoreceptor synapse. Human Molecular Genetics 15 (8): 1291–1302.
2. Blethrow, J. et al. (2007) Modular mass spectromic tool for analysis of composition and phosphorylation of protein complexes. PLoS ONE 2 (4): e358.
3. Catrein, I. et al. (2005) Experimental proof for a signal peptidase I like activity in Mycoplasma pneumoniae, but absence of a gene encoding a conserved bacterial type I
SPase. FEBS Journal 272: 2892–2900.
4. Cristea, I.M. et al. (2006) Tracking and elucidating alphavirus-host protein interactions. J Biol Chem 281: 30269–30278.
5. Drouet, J. et al. (2006) Interplay between Ku, Artemis and the DNA-dependent protein kinase catalytic subunit at DNA ends. J Biol Chem 281: 27784–27793.
6. Gudz, T.I. et al. (2006) Glutamate stimulates oligodendrocyte progenitor migration mediated via an alphav integrin/myelin proteolipid protein complex. J Neuroscience 26
(9) : 2458–2466.
7. Hara, T. et al. (2007) Mass spectrometry analysis of the native protein complex containing Actinin-4 in prostate cancer cells. Molecular and Cellular Proteomics 6: 479–491.
8. Harrison, M. et al. (2006) Some C. elegans class B synthetic multivulva proteins encode a conserved LIN-365 Rb-containing complex distinct from a NuRD-like complex. Proc
Natl Acad Sci, USA 103(45) : 16782–16787.
9. Hayes, M.J. et al. (2006) Early mitotic degradation of Nek2A depends on Cdc20-independent interaction with the APC/C. Nature Cell Biology 8(6) : 607–614.
10. Kantardzhieva, A. et al. (2006) MPP3 is recruited to the MPP5 protein scaffold at the retinal outer limiting membrane. FEBS Journal 273: 1152–1165.
11. Kawai, T. et al. (2006) Translational control of cytochrome c by RNA-binding proteins T1A-1 and HuR. Molecular and Cellular Biology 26 (8):3295–3307.
12. Maeda, Y. et al. (2006) PARP-2 interacts with TTF-1 and regulates expression of surfactant Protein-B. J Biol Chem 281 (14): 9600–9606.
13. Mazan-Mamczarz, K. et al. (2006) Translational repression by RNA-binding protein TIAR. Molecular and Cellular Biology 26(7): 2716–2727.
14. Ogawa, C. et al. (2007) Gemin2 plays an important role in stabilizing the survival of motor neuron complex. J Biol Chem 282 (15) : 11122–11134.
15. Pinsky, B.A. et al. (2006) Glc7/protein phosphatase 1 regulatory subunits can oppose the IPl1/Aurora protein kinase by redistributing Glc7. Molecular and Cellular Biology
26 (7) 2648–2660.
16. Reimers, K. et al. (2006) Sequence analysis shows that Lifeguard belongs to a new evolutionarily conserved cytoprotective family. Int J Molecular Medicine 18: 729–734.
17. Renvoisé, B. et al. (2005) Distinct domains of the spinal muscular atrophy protein SMN are required for targeting to Cajal bodies in mammalian cells. J Cell Science 119:
680–692
18. Schulze, W.X. et al. (2005) Phosphotyrosine interactome of the ErbB-receptor kinase family. Mol Syst Biol 1: 2005.0008.
19. Usui, K. et al. (2005) Protein-protein interactions of the hyperythermophilic archaeon Pyrococcus horikoshii OT3. Genome Biology 6:R98.
20. Wang, C.W. et al. (2006) Exomer: a coat complex for transport of select membrane proteins from the trans-Golgi network to the plasma membrane in yeast. J Cell Biology
174 (7): 973–983.
21. Zawacka-Pankau, J. et al. (2007) Protoporphyrin IX interacts with Wild-type p53 Protein in vitro and induces cell death of human colon cancer cells in a p53-dependent and
-independent manner. J Biol Chem 282 (4): 2466–2472.
22. Zissimopoulos, S. et al. (2007) Redox sensitivity of the ryanodine receptor interaction with FK506-binding protein. J Biol Chem 282 (10): 6976–6983.
www.invitrogen.com
©2007 Invitrogen Corporation. All rights reserved. These products may be covered by one or more Limited Use Label Licenses (see Invitrogen catalog or www.invitrogen.com). By use of these products you accept the
terms and conditions of all applicable Limited Use Label Licenses. For research use only. Not intended for any animal or human therapeutic or diagnostic use, unless otherwise stated. O-072624-r1 US 1107